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Mammalian Cell Assay Kit

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Matchmaker Mammalian Two-Hybrid System
In a Matchmaker mammalian two-hybrid assay (Figure 1), a bait protein is expressed as a fusion to the GAL4 DNA-binding domain (DNA-BD), while prey proteins are expressed as fusions to the VP16 activation domain of HSV. When a prey fusion protein interacts with the bait protein, the DNA-BD and AD bind to the PGAL4-E1b promoter to activate transcription of the secreted alkaline phosphatase reporter (SEAP). SEAP activity is detected using the Great EscAPe SEAP Chemiluminescence Kit 2.0.
Secreted Alkaline Phosphatase (SEAP) Reporter
SEAP is an excellent reporter for two-hybrid interactions and uses a simple, one-step, highly sensitive, chemiluminescent assay (Figure 2). Because SEAP is secreted into the growth medium, the signal is stable and can be left to accumulate so that even weak interactions can be assayed.
Figure 1. The mammalian two-hybrid principle. The bait protein is fused to the DNA binding domain from GAL4 and the prey protein is fused the transcriptional activation domain of VP16. If the two proteins interact at the PGAL4-E1b promoter (GAL promoter), SEAP is secreted into the growth medium.


Figure 2. Chemiluminescent detection of protein-protein interactions. The bait vector pM-53 expresses p53 fused to the GAL4 DNA-binding domain. When HEK 293 cells were cotransfected with this bait together with the SV40 large T antigen prey vector, pVP16-T, strong expression of SEAP was detected because large T antigen interacts with p53. The CP protein which does not interact strongly with p53, was used as a negative control.

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